Neurotransmitters are released at synapses by synaptic vesicles (SVs), which originate from SV precursors (SVPs) that have traveled along the axon. Because each synapse maintains a pool of SVs, only a small fraction of which are released, it has been thought that axonal transport of SVPs does not affect synaptic function. Here, studying the corticostriatal network both in microfluidic devices and in mice, we find that phosphorylation of the Huntingtin protein (HTT) increases axonal transport of SVPs and synaptic glutamate release by recruiting the kinesin motor KIF1A. In mice, constitutive HTT phosphorylation causes SV over-accumulation at synapses, increases the probability of SV release, and impairs motor skill learning on the rotating rod. Silencing KIF1A in these mice restored SV transport and motor skill learning to wild-type levels. Axonal SVP transport within the corticostriatal network thus influences synaptic plasticity and motor skill learning. Neurons communicate by transmitting chemical messages through their synapses. The number of synaptic vesicles (SVs) that carry these chemical messengers (neurotransmitters), the probability of the vesicles releasing neurotransmitter, and the vesicle quantal size, all affect synaptic strength and thus the ability to learn and remember . SVs actCaenorhabditis elegans, null mutants for the kinesin-related gene unc-104 or the vesicle-associated protein SAM-4 lead to defects in SVP transport, with a consequent lack of SVs at synapses and locomotor deficits , a protein that plays a prominent role in axonal transport , provideTo investigate whether HTT and its phosphorylation affect the transport of SVP in a physiologically relevant system, we reconstituted mature corticostriatal circuits in microfluidic devices . These dWe generated cortical and striatal neurons from embryos of wild-type (WT) mice and mice bearing either constitutively phosphorylated (HTT-SD mice) or unphosphorylatable HTT (HTT-SA mice) at embryonic day 15.5 (E15.5). HTT phosphorylation at serine 421 is mimicked by the replacement of the serine by an aspartic acid, which maintains the positive charge (S421D), whereas the unphosphorylatable form of HTT is obtained by mutating the serine into an alanine (S421A) . We tranConstitutive HTT phosphorylation (S421D) increased the anterograde velocity of VAMP2-positive vesicles , leadingWe next investigated whether this increased presynaptic anterograde transport of SVPs affects the capacity of presynaptic neurons to release glutamate from SVs at the corticostriatal synapses. We transduced presynaptic cortical neurons with a lentivirus encoding the indicator of vesicle release and recycling VGLUT1-pHluorin, thanks to the fusion of a pH-sensitive GFP (pHluorin) to the vesicular glutamate transport VGLUT1 . We thenWe had previously characterized HTT-SA, HTT-SD, and WT mice and found no differences between them in motor coordination , forelimTo better characterize this motor learning deficit, we examined the first and last days of training more closely. HTT-SD had greater difficulty adjusting to the rod initially than WT mice but improved by the end of the day . On day Motor skill learning relies on communication between the dorsal striatum and layer V pyramidal neurons in the motor cortex via the release of glutamate by the cortical afferences . We therThe lower facilitation in HTT-SD MSNs indicates that constitutive phosphorylation of HTT increases the probability of glutamate release in pyramidal cells. These findings are in agreement with the greater number of exocytic events in HTT-SD neurons within microfluidic devices .Since decreased facilitation could indicate changes in the number of SVs at the presynapse , we inveAlthough we did not observe less anterograde transport or preferential retrograde transport of VAMP2-mCherry in our experimental conditions , previouThe anterograde transport of SVP is driven predominantly by the kinesin-3 motor KIF1A . HTT andWe assessed this possibility by permeabilizing isolated axons within the distal part of microfluidic axonal compartments and, using Airyscan confocal high-resolution microscopy, we observed greater colocalization of HTT with KIF1A and VAMP2 in HTT-SD neuronal circuits than in WT . We thenWe next asked whether the phospho-HTT-mediated increase in SVP anterograde transport depends on KIF1A by using a validated sh-Kif1a . LentiviWe next measured VAMP2 transport in HTT-SD neurons and found greater anterograde velocity, number of anterograde vesicles, and positive net directional flux (and/or linear flow) than in WT neurons , confirmWe considered the possibility that the observed increase in SV release might be due in part to a synergistic action of BDNF at synapses, both because of the prominent role of HTT and its phosphorylation in regulating BDNF transport  and becaWe next investigated whether SVP anterograde transport via the HTT-KIF1A complex regulates the number of vesicles at synapses. We injected lentiviruses encoding either sh-scramble-GFP or sh-Kif1a-GFP into layer V of the HTT-SD motor cortex , whose nTo determine whether the modification in anterograde transport via the HTT-KIF1A complex within corticostriatal-projecting neurons is responsible for the defect in motor skill learning we observed in HTT-SD mice, we injected lentiviral vectors encoding sh-Scr-GFP and sh-Kif1a-GFP into 3-month-old WT and HTT-SD mice. Three weeks later, we subjected the mice to the same rotarod protocol as before . HTT-SD We show here that axonal transport of SVPs influences synaptic function. Specifically, HTT’s phosphorylation status fine-tunes SVP transport efficiency through its recruitment of KIF1A. Genetically blocking dephosphorylation at S421 impaired motor learning, and abolishing KIF1A activity in the context of constitutive phosphorylation only partially restored motor performance on the rotarod. Such a genetic approach is rather blunt compared to the sensitivity of (de)phosphorylation in responding to cellular signals, yet it enabled us to answer the question that motivated the study and show that axonal transport does influence synaptic homeostasis, with consequences for circuit function and behavior.Drosophila larval axons showed that reducing HTT levels decreases the transport of Rab3-positive vesicles  and PP2A . WhetherSeveral studies have linked a reduction in axonal anterograde transport of SVPs to a decline in synaptic function. Indeed, genetically impairing KIF1A reduces the number of SVs at nerve terminals and causes postnatal lethality . KIF1A lunc-104 that cause hyperactive axonal transport and abnormal accumulation of SVs  . Recent Altogether, our results highlight the importance of axonal SVP transport for synaptic transmission and identify a role for the HTT-KIF1A pathway as a regulator of SVP transport, synapse function, and motor skill learning within corticostriatal projecting neurons.Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Frédéric Saudou (frederic.saudou@inserm.fr).S421A/S421AHtt and S421D/S421DHtt mice  have been previously described (S421DorA/S421DorAHtt), or WT (+/+Htt) mice. Those mice were then used for electrophysiological and biochemical studies or processed to be imaged by electron microscopy. The number of animals was limited to the minimum number necessary per group in order to have at least an 80% chance of detecting a significant difference (power 1-β) and a risk of error α of 5%. This number was determined using a statistical test for estimating the optimal sample size using the variances determined in a preliminary study.escribed . They weFor biochemistry and neuronal culture (E15.5), the sex distinction of homozygous or WT mice was not made. Specific ages used for each experiment are indicated in the figure legends. C57BL/6J mice, purchased from Charles River Laboratory, were used for backcrosses to maintain the colony and to obtain WT E15.5 pups.6 cells in 120 µl). Cortical neurons were plated in the presynaptic chamber coated with poly-D-lysine (0.1 mg/ml) and striatal neurons were plated in the postsynaptic chamber coated with poly-D-lysin and laminin (10 µg/ml) with a final density of ~7000 cells/mm2. A growing medium was added to the synaptic chamber to equilibrate the flux. Neurons were left in the incubator for 2 hr and then all compartments were gently filled with growing medium. Neurons were cultured at 37°C in a 5% CO2 incubator for 10–12 days.Primary cortical and striatal neurons were dissected from E15.5 WT (C57Bl/6J) or HTT-SA or HTT-SD mouse embryos as previously described . They unBetween DIV 0 and DIV 4, cortical neurons within the presynaptic compartment were transduced with lentiviruses expressing VAMP2-mCherry, VGLUT1-pHluorin, or BDNF-mCherry. Neurons were washed out the next day. At DIV 8, cortical neurons were transduced with sh-Kif1a or sh-Scr lentiviruses.Motor skill learning was assessed using an accelerating rotarod . The tests were performed during the beginning of the light phase on male littermates housed in the same cages. Cages were transported to the experimental room at least 30 min before the tests to allow habituation of the mice to the room kept at a constant temperature (19–22°C) and humidity (40–50%). The day preceding the test, the mice were acclimated to the rod by one session (1 min at 4 rpm). Then, the accelerating rotarod assay was performed over 8 consecutive days with 10 sessions per day per mouse, increasing the speed from 4 to 40 rpm over 5 min. Each trial was separated by at least a 15 min resting period. The latency and the speed to fall off from the rotarod were recorded.Three-month-old HTT-SD and WT male mice were anesthetized by inhalation of isoflurane associated with a mix of oxygen and room air (3–5% of isoflurane for induction and 1–2% in the mask). The mouse head was then shaved and placed within the stereotaxic frame. The skin was incised, and the skull was bilaterally drilled. The capillary was inserted slowly. We injected the brains bilaterally at the following coordinates: anterior-posterior axis, AP, +1.54 mm; medial-lateral axis, ML, +1.6 mm or –1.6 mm; and dorsal-ventral axis, DV, -0.8 mm. 500nl of the diluted lentivirus  at 0.5µl/min speed using a nanoinjector. The capillary was slowly removed 1 min after the end of the injection to prevent the leak of the injected solution. The skull was then washed with saline solution, the skin was sutured and 1ml of NaCl 0.9% was injected subcutaneously. After surgery, mice were put alone in a warmed cage and monitored daily throughout recovery.Vglut1 cDNA sequence was amplified from an adult mouse brain. Its sequence from the 104th amino acid to the end was cloned after the pHluorin sequence in a Smal site in a superecliptic pHluorin containing vector used in GACCGGACCTTCTACCAGT) has already been published (GGGTGAACTCACGTCAGAA) (The VAMP2-mCherry construct was a kind gift from T Ryan’s laboratory. ublished . It has GTCAGAA) . It has GTCAGAA) , have beFor lentivirus production, all the plasmids were cloned into a pSin vector  by GatewMicrofluidic devices were generated as previously described . Briefly2.Videorecording of neurons plated in microfluidic devices was performed at DIV 12. Before recordings, DIV 12 neurons in the microchamber were carefully inspected and selected based on the absence of cell contamination. For double transductions (with sh-RNA lentiviruses), the transport of mCherry-tagged cargo was analyzed within GFP-positive axons. Images were acquired every 200 ms for 1 min on an inverted microscope  with X63 oil-immersion objective (1.46NA) coupled to a spinning-disk confocal system  connected to an electron-multiplying CCD (charge-coupled device) camera  at 37°C and 5% CO2. The same three fields per microchambers were acquired before and after a 4AP-bicuculline (respectively 2.5 mM and 50 µM) stimulation of the presynaptic chamber, four times in total (one before and three after stimulation).For the study of the exocytosis events, images were acquired every 200 ms for 1 min on an inverted microscope  with X63 oil-immersion objective (1.46NA) coupled to a spinning-disk confocal system  with TIRF microscopy  equipped with a camera Prime 95B sCMOS (Telelyne Photometrics) at 37°C and 5% CONeurons from the reconstituted corticostriatal network were fixed with a PFA/sucrose solution (4%/4% in PBS) for 20 min at room temperature (RT). After three washes of PBS, neurons were incubated first with a blocking solution  and then with primary antibodies for KIF1A , HTT , and mCherry  overnight at 4°C. The next day, neurons were washed three times with PBS followed by 1 hr incubation at RT of appropriate secondary antibodies (1:1000) and finally washed again three times with PBS. Images were acquired with a X63 oil-immersion objective (1.4 NA) using an inverted confocal microscope  coupled to an Airyscan detector. For 2D-STED microscopy, we used the Abberior kit containing the secondary antibodies  and coverslips were mounted with the Abberior mount solid. Images were taken with a 100× oil-immersion objective (1.46 NA) using the Abberior 2D-STEDYCON upright confocal microscope.For brain slices, brains were incubated in PFA 4% overnight and washed with PBS three times the next day. Then, brains were cut into 100-µm-thick slices using a vibratome. The slices were incubated with a blocking solution  for 2 hr at RT and then with antibody against GFP  overnight at 4°C. The day after, the primary antibody was removed by three washes of PBS before incubating the slices with the associated secondary antibody and finally with three washes of PBS. Finally, slices were incubated with DAPI (1/4000) for 15 min, washed three times with PBS, mounted on Superfrost slides by using Dako Faramount Aqueous Mounting Medium solution and coverslips. The slices were acquired with a ×10 objective (0.45 NA) using a slide scanner  and with a ×10 objective (0.3 NA) using an inverted confocal microscope  coupled to an Airyscan detector to improve signal-to-noise ratio and to increase the resolution.The NaveniFlex MR kit (Navinci #NF.MR.100) was used to study the interaction of endogenous HTT with endogenous KIF1A/VAMP2 in cortical neurons. The assay was performed following the manufacturer’s instructions. Primary antibodies were incubated with the same dilutions used for immunocytochemistry experiments. Images were acquired with a X63 oil-immersion objective (1.4 NA) using an inverted confocal microscope .Cortical neurons were plated in free culture, transduced at DIV 1 with sh-Kif1a or Sh-scr, and lysed at DIV 5 in NetN buffer  complemented with protease inhibitor cocktail (Roche).2, 5 mM EGTA, 0.5 mM D-glucose, 1.5 mM CaCl2, 20 mM DTT pH 7.2, protease inhibitor from Roche) on ice with a glass potter and then with a 25 G needle. Lysates were then centrifuged  and the supernatant, considered as the total fraction, is then centrifuged (3000 RPM for 10 min). The resulting supernatant was centrifuged . The supernatant was then ultracentrifuged  to obtain the vesicular fraction (the pellet) and the cytosolic fraction (the supernatant).A vesicular fraction from brains was prepared as described in All types of lysed samples were dosed by a Bradford reagent to quantify the protein concentration and then analyzed by SDS-PAGE transferred to PVDF membranes. Then, membranes were incubated for 45 min in a 5% BSA TBST  solution and incubated with primary antibodies against KIF1A , VAMP2 , pS421 , Vinculin , p150 , Tubulin  at 4°C, overnight. The next day, membranes were washed at least three times with TBST and incubated with secondary antibodies conjugated with horseradish peroxidase against mouse or rabbit (1:1000) for 2 hr at RT. Membranes were finally revealed with ECL (Thermo Scientific) after three washes of TBST.We anesthetized 3- to 4-month-old animals with 1 ml/kg of Doléthal and perfused them transcardially with cold PBS followed by 2% paraformaldehyde, 2% glutaraldehyde, and 0.1 M cacodylate cold solution. We removed brains from the skull and fixed them in a 0.1 M phosphate buffer pH 7.2 with 2% of glutaraldehyde and 2% of paraformaldehyde for 48 hr at 4°C before obtaining 2-mm-thick or 100-µm-thick slices from a mold and a vibratome, respectively. A 1 mm square piece of tissue was removed from the DLS; samples were then fixed again with the same solution for 72 hr, washed with phosphate buffer, and then post-fixed in a 0.1 M phosphate buffer pH 7.2 with 1% osmium tetroxide for 1 hr at 4°C. After extensive washes with water, samples were then stained with a solution of 1% uranyl acetate pH 4 in water for 1 hr at 4°C. They were further dehydrated through a gradient of ethanol (30%–60%–90% and three at 100%) and infiltrated with a solution of 1/1 epon/alcohol 100% for 1 hr and followed by several baths of fresh epon (Fukka) for 3 hr. The resin was then poured into capsules containing the samples, heated at 60°C for 72 hr for polymerization, and finally cut into ultrathin sections with an ultramicrotome (Leica). Sample sections were then post-stained with fresh solutions of 5% uranyl acetate and 0.4% of lead citrate, observed with a transmission electron microscope at 80 kV (JEOL 1200EX) and images were acquired with a digital camera . Analysis was performed with ImageJ and quantification of the number of synapses was performed on axon-free neuropil regions .2/95% O2-bubbled, ice-cold cutting solution containing (in mM): 125 NaCl, 2.5 KCl, 25 glucose, 25 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, and 1 pyruvic acid, and then transferred into the same solution at 34°C for 60 min and then moved to RT.All experiments were performed in accordance with the guidelines of the local animal welfare committee (Center for Interdisciplinary Research in Biology Ethics Committee) and the EU (directive 2010/63/EU). We prepared horizontal brain slices containing the somatosensory S2 cortex and the corresponding corticostriatal projection field in the dorsal striatum from mice (2–3 months of age) using a vibrating blade microtome . Brains were sliced in a 5% CO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, and 10 μM pyruvic acid, bubbled with 95% O2 and 5% CO2. Signals were amplified using EPC10-2 amplifiers . All recordings were performed at 34°C using a temperature control system  and slices were continuously superfused at 2–3 ml/min with the extracellular solution. Slices were visualized on an Olympus BX51WI microscope  using a 4×/0.13 objective for the placement of the stimulating electrode and a 40×/0.80 water-immersion objective for localizing cells for whole-cell recordings. The series resistance was not compensated. Recordings were sampled at 10 kHz, using the Patchmaster v2x32 program (HEKA Elektronik).For whole-cell patch-clamp recordings, borosilicate glass pipettes of 4–6 MΩ resistance contained (in mM): 105 K-gluconate, 30 KCl, 10 HEPES, 10 phosphocreatine, 4 ATP-Mg, 0.3 GTP-Na, 0.3 EGTA (adjusted to pH 7.35 with KOH). The composition of the extracellular solution was (in mM): 125 NaCl, 2.5 KCl, 25 glucose, 25 NaHCOFor paired-pulse protocols, electrical stimulations were performed with a bipolar electrode  placed in the layer V of the somatosensory S2 cortex. Electrical stimulations were monophasic at constant current . Currents were adjusted to evoke 50–200 pA EPSCs. Repetitive control stimuli were applied at 0.1 Hz. For each ISI, 20 successive EPSCs were individually measured and then averaged. Variation of input and series resistances above 20% led to the rejection of the experiment. Off-line analysis was performed with Fitmaster (Heka Elektronik). Statistical analysis was performed with Prism 5.02 software . All results are expressed as mean ± SEM. Statistical significance was assessed in non-parametric Mann-Whitney, one-sample t-tests using the indicated significance threshold (p).This analysis follows that of Vesicle velocity, directional flow, and vesicle number were measured on 100 µm of neurite using KymoTool Box ImageJ plugin, as previously described . AnterogFor each ISI, 20 successive EPSCs were individually measured and then averaged. Variation of input and series resistances above 20% led to the rejection of the experiment. Off-line analysis was performed with Fitmaster (Heka Elektronik). Statistical analysis was performed with Prism 5.02 software . All results are expressed as mean ± SEM. Statistical significance was assessed in non-parametric Mann-Whitney, one-sample t-tests using the indicated significance threshold (p).2 was normalized on the total number of nuclei (DAPI+).Immunostained vesicles in distal axons were quantified as previously shown . BrieflyThe same three fields per microchamber were acquired before and after a 4AP-bicuculline (respectively 2.5 mM and 50 µM) stimulation of the presynaptic chamber, four times in total (one before and three after stimulation). The movies were analyzed using a customized macro for ImageJ, thus the recording of the amplitude and the number of exocytosis events were automatized. The number of events was expressed as follows:whereyi expresses the difference of the number of events after  minus before  stimulation in HTT-SD neurons of the field i,j expresses the difference of the number of events after minus before stimulation in WT neurons of the field j and n of them have been averaged.xthe final value was normalized to 1, that is a given HTT-SD neuron field, whose activity after stimulation increased as much as that of the average of the WT neuron fields, will display a value of 1.The amplitude of the signal from stimulated neurons was normalized by that of the same neuron before stimulation.We used ImageJ to analyze synapse morphology. We counted the number of synapses in axon-free neuropil regions . SVs werStatistical calculations were performed using GraphPad Prism 6.0. Statistical parameters  are reported in the figure legends. For each dataset, we identified outliers using the ROUT test (Q=1%) and removed them from analysis. We performed a Shapiro-Wilk normality test with the threshold set at α=0.05; if the data followed a normal distribution, we used parametric tests, and if not, we used non-parametric tests. If we were analyzing two conditions we used a t-test (or a Mann-Whitney test if non-parametric). If comparing more than two conditions, we used a one-way ANOVA followed by Tukey’s post hoc analysis . If the datasets were interdependent, we used a two-way ANOVA followed by Tukey’s post hoc analysis if more than two groups are compared, or a Sidak’s post hoc analysis if only two groups are analyzed. For a nonlinear fit, we did a run test to determine whether the curve deviates systematically from the data. Low p value (ns) indicates that the curve poorly describes the data. *p<0.05; **p<0.01; ***p0.001; ****p<0.0001; ns, non-significant. This important study provides solid in vitro and in vivo data supporting the connection between Huntingtin's (HTT) phosphorylation state and the recruitment of Kif1A in the axonal anterograde trafficking of synaptic vesicles precursors. This works highlights the role of HTT-Kif1A synergy in modulating synaptic vesicle precursor transport and synaptic function, adding important information related to how synaptic vesicle pools are formed and replenished. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Suzanne Pfeffer as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Huntingtin-KIF1A-mediated axonal transport of synaptic vesicle precursors influences synaptic transmission and motor skill learning in mice" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1. The authors conclude that there is a change in synaptic transmission based on subtle modulation of total synaptic vesicle number. However, it is not very well supported, and the literature argues against total vesicle number being a major point of control for neurotransmission. The authors should discuss this point and take into account the direct effect on vesicle exocytosis or short-term synaptic plasticity.2. Figure 1A-B would be strengthened by proximity ligation assays showing the extent of endogenous interaction between huntingtin and VAMP2 in axons of WT versus HTT-SD and HTT-SA mutants. Perhaps this can be incorporated in figure 5B-C, where to this reviewer's opinion, a PLA approach would be better to show and quantify the interaction between KIF1A and HTT (see the separate point). Alternatively, a PLA for KIF1A and HTT combined with IF for VAMP could work as well.3. Figure 1C – How do the authors explain the high variance in the HTT-SA mutant? It seems that it could reach significance with 1-2 more devices. Why do they think they do not see reduced synaptic release but a trend toward increased one? Representative movies would help understand the system.4. Figure 4 – Do the authors detect a change also in synapse size? The synaptic vesicle count is not normalized to synapse area/size/volume.5. Figure 5 – The images in figures 5B-C are challenging to interpret and, therefore, also fail to represent the data. We don't see much colocalization. VAMP2 does not seem to colocalize with Kif1A, while it seems to overlap better with HTT. Could the authors provide a quantification of Kif1a-VAMP2 and HTT-VAMP2 colocalization and discuss the implications? As mentioned before, a proximity ligation assay would be suitable for strengthening this point. Also, since this experiment is done on fixed cells, the authors should use cells that were not-infected with VAMP2-mCherry cells but rather observe the endogenous VAMP2 colocalization in KIF1A and HTT. It will be nice to show also VAMP2 levels in 5D-E.6. The statistical approach should be better substantiated. The authors often make use of the Man Whitney test, which as far as I know is non-parametric and does not assume normal distribution. However, the data presented are always averages with standard error of the means (s.e.m.). Seems to me to be a contradiction. The authors should comment on why they chose this test over a parametric one and, if their dataset is not normally distributed, maybe they should check if their representation is correct. In addition, ANOVA is used with different post-tests. Most of the time Dunn is used, but also Sidak and Tuckey are used. I could not infer from the dataset why that is and maybe the authors could explain.– In Figure 1C the significance was determined using a t-test? ANOVA seems more appropriate. Which statistical assay was used in Figure 3?– Figure 1A and B. Is the comparison between HTT-SD and HTT-SA significant?– Figure 2B. All the latency to fall of all the mice in all the repeat for all the days tested have been pulled together? If this is case, this representation of the rotarod data is unusual, please explain what the advantage is compared to just showing the curve of the entire experiment as done in panel C.– Figure 3C. The quantification has an abundance of asterisks. It is unclear what was compared with what.– Fig S2. Why are HTT-SD and HTT-SA separately compared to the wild type control? They should be grouped together in the same chart and ANOVA should be used.7. Why the authors do not elaborate more on the difference or lack thereof between HTT-SD and HTT-SA and they are so quick to drop the HTT-SA mutant from the rest of study? The alanine mutant are expected to be non phosphorylatable and thus should present a more drastic difference in relation to the constitutive phosphorylated mutant one (SD). The wild type HTT, which can still be phosphorylated, should be somewhere in the middle. This seems counterintuitive. Have the authors tested for the phosphorylation status of their mutants?8. It seems counterintuitive that HTT-SD promotes SVPs transport, but results in a behavioral deficit. Although the authors provide the explanation of these results, but it would be helpful if the authors could provide more evidence. For example, the hypothesis of an increase in docked SVs could be tested. The authors provide already EM images . Could they quantify the number of docked vesicles? If this is difficult, maybe the number of vesicles adjacent to the membrane?Reviewer #1 (Recommendations for the authors):I find this manuscript suitable for publication following revisions and corrections as detailed below.1. Figure 1A-B would be strengthened by proximity ligation assays showing the extent of endogenous interaction between huntingtin and VAMP2 in axons of WT versus HTT-SD and HTT-SA mutants. Perhaps this can be incorporated in figure 5B-C, where to this reviewer's opinion, a PLA approach would be better to show and quantify the interaction between KIF1A and HTT (see the separate point). Alternatively, a PLA for KIF1A and HTT combined with IF for VAMP could work as well.2. Figure 1C – How do the authors explain the high variance in the HTT-SA mutant? It seems that it could reach significance with 1-2 more devices. Why do they think they do not see reduced synaptic release but a trend toward increased one? Representative movies would help understand the system.3. Figure 4 – Do the authors detect a change also in synapse size? The synaptic vesicle count is not normalized to synapse area/size/volume.4. Figure 5 – The images in figures 5B-C are challenging to interpret and, therefore, also fail to represent the data. As mentioned before, a proximity ligation assay would be suitable for strengthening this point. Also, since this experiment is done on fixed cells, the authors should use cells that were not-infected with VAMP2-mCherry cells but rather observe the endogenous VAMP2 colocalization in KIF1A and HTT. It will be nice to show also VAMP2 levels in 5D-E.Reviewer #2 (Recommendations for the authors):The manuscript presents a solid set of data, which are in my opinion broadly supportive of the authors conclusions. A lot of work has gone into the incorporation of multiple approaches. I have a set of minor considerations that I hope might be helpful to the authors.1) The statistical approach should be better substantiated. The authors often make use of the Man Whitney test, which as far as I know is non-parametric and does not assume normal distribution. However, the data presented are always averages with standard error of the means (s.e.m.). Seems to me to be a contradiction. The authors should comment why they chose this test over a parametric one and, if their dataset is not normally distributed, maybe they should check if their representation is correct. In addition, ANOVA is used with different post-tests. Most of the time Dunn is used, but also Sidak and Tuckey are used. I could not infer from the dataset why that is and maybe the authors could explain.In Figure 1C the significance was determined using a t-test? ANOVA seems more appropriate. Which statistical assay was used in Figure 3?2) Figure 1A and B. Is the comparison between HTT-SD and HTT-SA significant?3) Figure 2B. I am not sure if I interpret this chart correctly. All the latency to fall of all the mice in all the repeat for all the days tested have been pulled together? If this is case, I am not sure I have encountered this representation of the rotarod data before and I wonder what the advantage is compared to just showing the curve of the entire experiment as done in panel C.4) Figure 3C. The quantification has an abundance of asterisks. I could not figure out what was compared with what. I wonder if the comparisons could be expressed more clearly.5) Figure 5C. Maybe the author could find another image. I cannot see much colocalization. VAMP2 does not seem to colocalize with Kif1A, while it seems to overlap better with HTT. Could the authors provide a quantification of Kif1a-VAMP2 and HTT-VAMP2 colocalization and discuss the implications?6) Fig S2. Why are HTT-SD and HTT-SA separately compared to the wild type control? They should be grouped together in the same chart and ANOVA should be used.7) Following from point 6 and 2, I am not sure why the authors do not elaborate more on the difference or lack thereof between HTT-SD and HTT-SA and they are so quick to drop the HTT-SA mutant from the rest of study. I expected that the alanine mutant would be non phosphorylatable and thus should present a more drastic difference in relation to the constitutive phosphorylated mutant one (SD). The wild type HTT, which can still be phosphorylated, should be somewhere in the middle. This seems counterintuitive. Have the authors tested for the phosphorylation status of their mutants?8) Seems counterintuitive that HTT-SD promotes SVPs transport, but results in a behavioral deficit. I understand the explanation provided, but I wonder if the authors could provide more evidence. For example, the hypothesis of an increase in docked SVs could be tested. The authors provide already EM images . Could they quantify the number of docked vesicles? If this is difficult, maybe the number of vesicles adjacent to the membrane? Essential revisions:1. The authors conclude that there is a change in synaptic transmission based on subtle modulation of total synaptic vesicle number. However, it is not very well supported, and the literature argues against total vesicle number being a major point of control for neurotransmission. The authors should discuss this point and take into account the direct effect on vesicle exocytosis or short-term synaptic plasticity.There are two issues here: the reviewer is not convinced by our data that synaptic transmission is affected by subtle changes in vesicle number, and believes the literature argues against vesicle number having much influence on neurotransmission. We'll take each issue in turn.We decided to analyze the distribution of vesicles in the presynaptic compartment. We divided the PSD into three 40 nm-wide zones: the proximal, active zone 1 (0 to 40 nm); zone 2 (40 to 80 nm); and the distal zone 3 (80 to 120 nm). Although vesicle pools are not anatomically segregated , the most proximal zone is likely to contain the readily releasable pool (RRP) while the most distal zone is likely to be enriched in vesicles from the reserve pool (RP) . WT and HTT-SD synapses did not differ in these last 120 µm of the axon terminal area , but HTT-SD presynapses contained significantly more vesicles in zone 3 . This correlates with the increased anterograde axonal transport leading to an accumulation of vesicles at the synapse, adding to the already-filled reserve pool.Drosophila, reduced recruitment of SVs from the reserve pool to the RRP impairs short-term synaptic plasticity and memory . While location relative to the membrane does not reliably define vesicular pool identity , our results are in agreement with the increased anterograde axonal transport of SVP we observed in vitro in HTT-SD neurons. Indeed, it is expected that vesicles are likely to accumulate mostly where the SVPs detach from the KIF1A motor, a location which is away from the membrane and the active zone . In addition, newly produced vesicles arriving at the presynaptic zone are preferentially released . Since all the vesicle pools are dynamic and interdependent , this study suggests that modifying the distal pool of vesicles would affect this dynamic instead of the number of docked vesicles adjacent to the membrane. This hypothesis is supported by the fact that modulating the size or dynamicity [?] of the reserve pool affects plasticity and memory .The literature supports our contention that this greater number of vesicles in the distal pool might influence the replenishment of the RRP  and/or the recycling pool . Several studies have linked the reserve pool to neurotransmission: In These findings are introduced in the Results section and now addressed in the discussion.2. Figure 1A-B would be strengthened by proximity ligation assays showing the extent of endogenous interaction between huntingtin and VAMP2 in axons of WT versus HTT-SD and HTT-SA mutants. Perhaps this can be incorporated in figure 5B-C, where to this reviewer's opinion, a PLA approach would be better to show and quantify the interaction between KIF1A and HTT (see the separate point). Alternatively, a PLA for KIF1A and HTT combined with IF for VAMP could work as well.Good suggestion. We now provide PLA experiments and show a significantly increased association of HTT with KIF1A in HTT-SD neurons . We were unsuccessful in combining IF for VAMP with PLA for KIF1A and HTT, but we measured association of KIF1A or HTT to VAMP2 vesicles in HTT-SD neurons using PLA  and Airyscan microscopy . These latter studies did not find consistent changes in the association of HTT or KIF1A to VAMP2. See also our response under point 5 below.3. Figure 1C – How do the authors explain the high variance in the HTT-SA mutant? It seems that it could reach significance with 1-2 more devices. Why do they think they do not see reduced synaptic release but a trend toward increased one? Representative movies would help understand the system.This question prompted us to re-analyze the original data and plot all the regions of interest (ROI) values rather than their mean as we did before. We analyzed 6712 events for WT neurons, 4640 events for the SD neurons and 5176 for the SA neurons within 60 seconds. This increased the statistical power and, because the data do not follow a normal distribution, we presented the data as a box-whisker plot . Although one point is particularly low (but was not an outlier), the distribution is now seen to be very similar to the one in HTT-SD condition. There does not appear to be a trend toward increased synaptic release.To help the reader to better understand our experimental set up, we now provide one movie (after stimulation) in which the distal part of the axonal channels can be seen in the upper part of the frame, the synaptic chamber in the middle and the distal part of the dendritic channels in the lower part of the frame. Arrows indicate transient spots of exocytosis that occur in the synaptic compartment and that have been quantified using a customized macro for Image, as described now in the Material and methods section (new Video 1).4. Figure 4 – Do the authors detect a change also in synapse size? The synaptic vesicle count is not normalized to synapse area/size/volume.Excellent point. We analyzed the images obtained from electron microscopy and found that indeed, HTT-SD presynapses are smaller than WT . Normalizing the synaptic vesicle count to synapse size confirms that HTT-SD synapses have more vesicles than WT synapses .5. Figure 5 – The images in figures 5B-C are challenging to interpret and, therefore, also fail to represent the data. We don't see much colocalization. VAMP2 does not seem to colocalize with Kif1A, while it seems to overlap better with HTT. Could the authors provide a quantification of Kif1a-VAMP2 and HTT-VAMP2 colocalization and discuss the implications? As mentioned before, a proximity ligation assay would be suitable for strengthening this point. Also, since this experiment is done on fixed cells, the authors should use cells that were not-infected with VAMP2-mCherry cells but rather observe the endogenous VAMP2 colocalization in KIF1A and HTT. It will be nice to show also VAMP2 levels in 5D-E.As explained above under point 2, we now provide PLA experiments demonstrating a specific interaction between HTT and KIF1A and a statistically significant increased association of HTT with KIF1A in HTT-SD neurons . We also performed high-resolution Airy Scan confocal experiments to quantify HTT interaction with KIF1A  and found a significant increase in the interaction between HTT and KIF1A in HTT-SD neurons.As requested, we also performed PLA experiments on endogenous VAMP2 colocalization with KIF1A and HTT , but we did not observe significant changes in the association of HTT or KIF1A to endogenous VAMP2. Using Airy Scan confocal microscopy  we observed similar association of HTT or KIF1A to VAMP2-mCherry. This suggests that most of the effect induced by HTT phosphorylation is through the increased interaction between motors (KIF1A) and HTT. These findings on the localization of HTT on vesicles independently of its phosphorylation at S421 is in agreement with our previous studies . Finally, we also show VAMP2 levels .Together with our proteomic data, our results obtained with 2D-STED microscopy, PLA, Airy Scan microscopy, and biochemical analyses of synaptic vesicle preparation  provide strong evidence that KIF1A interacts with HTT on VAMP2 immuno-positive vesicles and that the HTT-KIF1A interaction is enhanced in HTT-SD neurons.6. The statistical approach should be better substantiated. The authors often make use of the Man Whitney test, which as far as I know is non-parametric and does not assume normal distribution. However, the data presented are always averages with standard error of the means (s.e.m.). Seems to me to be a contradiction. The authors should comment on why they chose this test over a parametric one and, if their dataset is not normally distributed, maybe they should check if their representation is correct.We apologize for not better describing the statistics, and we now provide more information in both the Methods and the figure legends. We checked the normal distribution with a Shapiro-Wilk test. In most of the cases, our data sets did not follow a normal distribution. This is the reason why we chose a non-parametric test to compare 2 conditions. Then, as suggested by the reviewers, we modified the representation of the data that were analyzed by a Mann-Whitney test and now show box-whisker plots .In addition, ANOVA is used with different post-tests. Most of the time Dunn is used, but also Sidak and Tuckey are used. I could not infer from the dataset why that is and maybe the authors could explain.As we now explain in the Methods, for parametric data we used one way ANOVA followed by a Tukey test; for nonparametric data, we used one way ANOVA followed by Dunn. When comparing at least three groups we used two-way ANOVA followed by Holm-Sidak, which has a better capacity than Tukey to discriminate potential significant differences. – In Figure 1C the significance was determined using a t-test? ANOVA seems more appropriate. Which statistical assay was used in Figure 3?For greater statistical accuracy, we used all data points instead of the average of three regions of interest. We then used a non-parametric one-way ANOVA followed by a Dunn’s pot hoc test .The test used in Figure 3 was described: “All results are expressed as mean ± SEM. Statistical significance was assessed in non-parametric Mann Whitney, one-sample t-tests using the indicated significance threshold (p)” When comparing each result (comparing the different conditions that correspond to single column) vs the value 1 (dotted line) we used one-sample t-tests. We removed the other statistical comparisons that were misleading because unnecessary.– Figure 1A and B. Is the comparison between HTT-SD and HTT-SA significant?Since we do not know the physiological level of phosphorylation in our experiment, we compared constitutive phosphorylation and absence of phosphorylation to the wild type condition. Consequently, we limited the number of post-hoc tests (to WT vs. HTT-SD and WT vs. HTT-SA only) to avoid loss of significance upon multiple comparisons. In our experimental conditions, the physiological level of HTT phosphorylation is rather low, which explains why we observe a significant difference in HTT-SD neurons but not in HTT-SA neurons. We mention this in the Discussion.– Figure 2B. All the latency to fall of all the mice in all the repeat for all the days tested have been pulled together? If this is case, this representation of the rotarod data is unusual, please explain what the advantage is compared to just showing the curve of the entire experiment as done in panel C.This is a valid point. We have modified the figure and show the curve of the entire experiment as done in panel C. We made the same modifications for the new Figure 8B, C.– Figure 3C. The quantification has an abundance of asterisks. It is unclear what was compared with what.Our intent was to show all the statistical differences but the asterisks did overburden the figure.We now provide a simpler representation of the data with an emphasis on the key results: comparisons of each condition to the value 1.– Fig S2. Why are HTT-SD and HTT-SA separately compared to the wild type control? They should be grouped together in the same chart and ANOVA should be used.HttS421D/+ x HttS421D/+ crosses on one hand and HttS421A/+ x HttS421A/+ on the other). Consequently, the WT mice originate from different strains and cannot be grouped.We separated the two genotypes because we compared each of them to their respective WT littermates, which were obtained from separate breedings . The wild type HTT, which can still be phosphorylated, should be somewhere in the middle. This seems counterintuitive.We initially focused on the HTT-SD mice because the effects on axonal transport of vesicles, their release at the synapse, and behavior were clearer. We have now added more data on HTT-SA mice. These new experiments indicate subtle alterations in the HTT-SA condition, which, although less marked, are the opposite of the alterations observed when HTT is consistently phosphorylated. We mention these points in the Discussion.Have the authors tested for the phosphorylation status of their mutants?When S421 is mutated into aspartic acid (mimicking constitutive phosphorylation) or into unphosphorylatable alanine, HTT is no longer recognized by anti-S421 phospho-HTT antibodies. See  and new Supplementary Figure S5B.8. It seems counterintuitive that HTT-SD promotes SVPs transport, but results in a behavioral deficit. Although the authors provide the explanation of these results, but it would be helpful if the authors could provide more evidence. For example, the hypothesis of an increase in docked SVs could be tested. The authors provide already EM images . Could they quantify the number of docked vesicles? If this is difficult, maybe the number of vesicles adjacent to the membrane?We were not able to quantify the number of docked vesicles but, as noted under points 1 and 4, we counted the number of vesicles at different distances from the membrane . We found no difference in terms of the density of vesicles within the zone adjacent to the membrane in HTT-SD axon terminals. These results suggest that what changes in HTT-SD mice is not the number of docked vesicles at the presynapse but rather the density of the pool of vesicles distal from the membrane that is increased. While we cannot confirm the identity of this pool—location relative to the membrane does not constitute a reliable technique to define vesicular pool identities —these results are in agreement with the increased anterograde axonal transport of SVP we observed in HTT-SD neurons in vitro.These findings are introduced in the Results section lines 183-204 and discussed in lines 377397.ReferencesDrosophila Fragile X Syndrome and Hereditary Spastic Paraplegias. Front Mol Neurosci, 9, 60. doi:10.3389/fnmol.2016.00060Bodaleo, F. J., and Gonzalez-Billault, C. (2016). 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The correlation between the two equations was almost perfect (rho 0.997) but decreased when the directly determined data were compared to those calculated with Sampson’s equation (rho 0.954) or Friedewald’s method (rho 0.939). Conclusion: direct determination generated higher values of LDL-C concentration through a probable systematic overestimation.Background: low-density lipoprotein cholesterol (LDL-C) is a strong cardiovascular risk factor, but the methods used to correctly determine it are is still questioned. The aim of this study was to compare the direct determination of LDL-C levels, obtained with the Roche cobas c system, with LDL-C values calculated through Sampson’s and Friedewald’s equations in very high-risk patients with triglycerides concentrations of less than 2.25 mmol/L (<200 mg/dL). Methods: in 127 consecutive patients with a recent diagnosis of acute coronary syndrome and triglycerides of less than 2.25 mmol/L, plasma LDL-C was measured directly and calculated with Sampson’s and Friedewald’s equations before hospital discharge, and the data were compared. Results: median LDL values obtained with the Friedenwald and Sampson equations and with direct determination were 2.31 (IQR 1.59–3.21), 2.36 (IQR 1.66–3.26) and 2.64 (IQR 1.97–3.65) mmol/L, respectively. Direct measurements were higher by 0.35 and 0.40 mmol/L when compared to the levels calculated with Sampson’s or Friedewald’s equations, respectively ( Cardiovascular disease (CVD), especially atherosclerotic cardiovascular disease (ASCVD), is responsible for nearly 4 million deaths yearly in Europe, accounting for more than 60 million potential years of life lost to CVD annually . It is aTherefore, the latest European guidelines on Dyslipidaemias have once more stressed the importance of ASCVD prevention by promoting healthy lifestyle behaviour, with strong reductions in cardiovascular (CV) risk factors, such as low-density lipoprotein cholesterol (LDL-C) or blood pressure (BP), diabetes, and smoking status . NumerouWith these rigorous recommendations, it is very important to standardize LDL-C determinations, also taking into consideration the fact that lipoprotein cholesterol concentrations differ significantly when measured in fed or in fasted subjects  and thatIt is also mandatory to entrust a reliable laboratory method for lipid and lipoproteins estimation to guide therapeutic decisions, especially in patients on the recent most effective lipid-lowering therapy, such as proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, which can markedly decrease LDL-C concentration .The β-quantification is considered the gold standard for LDL-C measurement, but it is time consuming and expensive and almost never used in daily clinical practice. Surrogate methods are indirect determinations obtained through formulas that extrapolate the LDL-C from other lipid parameters. The well-known Friedewald formula  is used Martin et al. ,11 develRecently, Sampson et al.  developeTo overcome the problems associated with the calculation of LDL-C, in recent years, much effort has been put into the development of tests for the direct measurement of LDL-C (dLDL-C) ,17, whicThe aim of our study was to compare direct LDL-C concentration determination to data calculated using the new Sampson’s equation and Friedewald formula in very-high-risk patients with TG levels of less than 2.25 mmol/L (200 mg/dL) in order to highlight possible differences that should be known and taken in account in clinical practice. In this observational study, LDL-C concentrations were determined and calculated in 130 consecutive patients discharged with the diagnosis of acute coronary syndrome from the Department of Cardiology of the San Maurizio Regional Hospital of Bolzano, Italy, during the 4-month period between 1 February and 31 May 2018. All the patients had undergone a percutaneous coronary intervention with complete revascularisation during their hospital stay. They were then followed up in our outpatient coronary clinic during the subsequent six months. Having already experienced an acute coronary syndrome, all these patients were considered to have very high cardiovascular risk and put on a combination of high-efficacy statins, ezetimibe or PCSK9 inhibitors. Written informed consent for the evaluation of the clinical data accrued during the admission was obtained from all patients, and the study was conducted according to the latest version of the Declaration of Helsinki given by the World Medical Association. Formal approval of the protocol by the local Ethics Committee was considered unnecessary because the study did not interfere with the usual clinical routine, as the assessment of lipid status was part of the routine examinations prescribed to patients after the acute event, and the data were handed anonymously. In all patients, plasma LDL-C was measured before hospital discharge with LDL-Cholesterol Gen.3 , an in vitro test for the quantitative determination of LDL-cholesterol in human serum and plasma on Roche/Hitachi cobas c systems (Roche Diagnostics). LDL-Cholesterol Gen.3 is a homogeneous enzymatic colorimetric assay in which cholesterol esters and free cholesterol in LDL are measured on the basis of a cholesterol enzymatic method using cholesterol esterase and cholesterol oxidase in the presence of surfactants that selectively solubilize only LDL; surfactants and a sugar compound contained in the assay inhibit the enzyme reactions to the lipoproteins other than LDL, and, for this reason, the cholesterol contained in HDL, VLDL and chylomicron is not determined . All measurements were performed on the Roche cobas c 502 analyser following the manufacturer’s instructions.Aside from this direct determination, LDL-C was also calculated using the Sampson equation and Friedewald formula (reported below), and the results obtained with the three methods were compared to each other in 127 of the 130 patients. Three patients were excluded from the analysis because they had triglycerides concentration of more than 2.25 mmol/L (200 mg/dL), therefore, theoretically leading to some inaccuracy in the Friedewald formula. The choice of excluding patients with higher triglyceride values was made in order to avoid even minimal interference biases in the Friedewald formula.p-value of less than 0.05 was considered statistically significant. Bland and Altman plots [Continuous variables were described using the range, means and standard deviations if normally distributed and medians and interquartile ranges if not. The departure from normality of the LDL-C levels was assessed using the skewness and kurtosis tests for normality . Data on LDL-C were inspected using scatter plots and adding both linear and non-linear fitting functions. The agreement between different LDL-C values obtained in the same patients with the two different equations and direct determination was assessed using Lin’s concordance correlation coefficient for agreement rho on a continuous measure ,21, whican plots  were genThe clinical and demographic characteristics of the patients enrolled are reported in p < 0.001 for all comparison).The median LDL-C values obtained with the Friedenwald and Sampson equations and with direct determination were 2.31 (IQR 1.59–3.21), 2.36 (IQR 1.66–3.26) and 2.64 (IQR 1.97–3.65) mmol/L (89.4 (IQR 61.4–124.0), 91.2 (IQR 64.2–126.1) and 102.0 (IQR 76.2–141.0) mg/dL), respectively. Direct LDL-C measurements were higher by 0.35 (IQR 0.25–0.45) and 0.40 (IQR 0.29–0.49) mmol/L (13.4 (IQR 9.7–17.4) and 15.6 (IQR 11.2–19.2) mg/dL) when compared to the levels calculated with Sampson’s or Friedewald’s equations, respectively , with a 50% reduction in baseline values. The resulting LDL-C values were already inferior to 1.4 mmol/L in 18 and 20 patients using Sampson’s and Friedewald’s equations, respectively, and only in 12 when LDL-C was measured directly with the Roche system.The Bland–Altmann plot comparing LDL-C directly measured with the results calculated using Friedewald’s equation showed a negative bias, indicating higher values for the direct determination and a mean difference of −0.39 mmol/L (−15.3 mg/dL) . A similar negative bias was also found with Sampson’s equation, showing higher values for direct determination and a mean difference of −0.34 mmol/L (−13.2 mg/dL) , whilst Direct LDL-C measurement (variable y) was linearly related (Cusum test n.s.) to the data obtained with Friedewald’s formula (variable x) according to Passing-Bablok regression as y = 9.048 (95% CI 7.019 to 11.330) + 1.0656 (95% CI 1.043 to 1.092) X. A similar relation was found between direct LDL-C measurement and the data obtained with Sampson’s equation (variable z): y = 7.924 (95% CI 5.759 to 10.209) + 1.059 (95% CI 1.037 to 1.081) Z. The Passing-Bablok regressions indicate constant and proportional differences between the measured and calculated value of LDL-C with both equations, whilst the data derived from both equations showed good agreement. The correlation between the data obtained with the two equations  was almost perfect (rho 0.997). It decreased to substantial (rho 0.954) when the directly determined data were compared to those calculated using Sampson’s equation and only to moderate (rho 0.939) when the direct determination was compared to data obtained with Friedewald’s method A,B. Our results confirm previous observations of less reliability in terms of direct LDL-C determination when compared to Friedewald or Sampson equations in very-high-risk patients with TG levels of less than 2.2 mmol/L (<200 mg/dL), in whom both formulae have been considered very accurate . Direct Very important clinical decisions are routinely taken concerning the LDL-C values provided by clinical laboratories, which are often estimated with the Friedewald formula. This calculation is not reliable if the patient is not fasting or if the serum TG values are >4.5 mmol/L (>400 mg/dL) , but it The LDL-C reference method would require the ultracentrifugation of the plasma for 18 h at its own density (1.006 g/mL), the measurement of cholesterol in the 1.006 g/mL infranatant fraction and the subtraction of HDL-C from this value to obtain the LDL-C concentration . This meIn some studies, calculated LDL-C and direct LDL-C showed very strong correlations, but calculated LDL-C can underestimate LDL-C for TG >177 mg/dL (2 mmol/L) and may be misleading at very low LDL-C levels, especially in the presence of high TG ,26,27,28The latest American guidelines on the management of blood cholesterol recommend the measurement of direct LDL-C or modified LDL-C estimates with the Martin equation in class IIa to improve accuracy over the Friedewald formula in patients with LDL-C levels less than <1.8 mmol/L (<70 mg/dL) ,30. No rDifferently, direct HDL-C assays are universally performed by routine clinical laboratories because the test is fully automated and thus avoids the labor-intensive manual precipitation step used to remove LDL before measuring HDL-C . MeasuriThe new Sampson equation should be more reliable when compared to the Friedewald formula, especially in the case of high TG levels , but ourDifferent from the American guidelines, the latest European guidelines on dyslipidaemias state that plasma LDL-C can be either calculated or measured directly . SeveralOther indexes, such the triglyceride–glucose index (TyG), have been recently proposed for a more exhaustive risk classification in specific populations, such as patients with diabetes and acute coronary syndrome . The TyGIn our study, the direct determination with the Roche system generated higher values of LDL-C when compared to the data obtained through the Friedewald equations in patients with low TG, where it has traditionally proven excellent accuracy. The Sampson equation confirmed the reliability of the Friedewald formula, leading us to hypothesize a systematic overestimation of direct determination, even if it should be kept in mind that formulas are not the gold standard but only the best approximation known. This discrepancy should also be taken into consideration in registries on lipid control, as in the recently published SANTORINI , where sThe optimal management of patients requiring lipid-lowering therapy should be based on reliable laboratory methods, particularly in high- or very-high-risk patients. It is, therefore, essential to rely on a validated method as much as possible, reflecting the gold standard process. As recently reported by Choi et al., in order to accurately evaluate LDL-C, a laboratory-specific equation together with direct determination is needed . PhysiciIt is always necessary to know the limits of the method used in our laboratory for LDL-C determination because a wrong evaluation of the lipid profile can mislead physicians. In our case, being aware of the possible systematic overestimation of the Roche system could suggest preferring indirect calculation through Friedenwald or Sampson formulas.The main limit of our observations is the small number of the patients included, which is partially counterbalanced by the very strict selection of the population.Our study shows that direct determination generated higher values of LDL-C concentration through a probable systematically overestimation when compared to values obtained through Sampson’s and Friedewald’s equations, which showed an excellent reliability in patients within very-high-risk patients and TG levels of less than 2.2 mmol/L (<200 mg/dL). It is always necessary to be well aware of the limits of the method used in our laboratory for LDL-C determination in order to avoid misclassification errors, which could lead to inappropriate therapy. Even international studies or registries on lipid-lowering therapy should take into consideration the possible discrepancies deriving from different methods used in LDL-C evaluation.


Chitosan exhibits unique properties making it a suitable material for drug delivery. Considering the rising popularity of hydrogels in this field, this work offers a comprehensive study of hydrogels constituted by chitosan and cross-linked with 1,3,5-benzene tricarboxylic acid . Hydrogels were prepared by cross-linking chitosan with BTC in different concentrations. The nature of the gels was studied through oscillatory amplitude strain and frequency sweep tests within the linear viscoelastic region (LVE) limit. The flow curves of the gels revealed shear thinning behavior. High G′ values imply strong cross-linking with improved stability. The rheological tests revealed that the strength of the hydrogel network increased with the cross-linking degree. Hardness, cohesiveness, adhesiveness, compressibility, and elasticity of the gels were determined using a texture analyzer. The scanning electron microscopy (SEM) data of the cross-linked hydrogels showed distinctive pores with a pore size increasing according to increasing concentrations (pore size range between 3–18 µm). Computational analysis was performed by docking simulations between chitosan and BTC. Drug release studies employing 5-fluorouracil (5-FU) yielded a more sustained release profile with 35 to 50% release among the formulations studied in a 3 h period. Overall, this work demonstrated that the presence of BTC as cross-linker leads to satisfactory mechanical properties of the chitosan hydrogel, suggesting potential applications in the sustained release of cancer therapeutics. Hydrogels are cross-linked networks and hold hydrophilic functional groups in their structure. Hydrophilic functional groups, such as amine , synthenetworks , polyvinnetworks , polyacrnetworks , and polnetworks ) or seminetworks . The polnetworks . These fnetworks .β-(1 → 4)-2-acetamido-D-glucose and β-(1 → 4)-2-amino-D-glucose units and formed via the process of deacetylation of chitin. The chemical structure of chitosan is composed of two main sugars, namely glucosamine and N-acetyl glucosamine , which account for its solubility [At physiological pH, chitosan acts as a mucoadhesive polymer. Here, the amino groups become protonated and act as a determining factor in chitosan’s mucoadhesive properties . When dilubility  and its lubility . 2) of chitosan. In addition, possible interactions, such as ionic, hydrogen, and π-π bonding, were reported in the literature [Chitosan is in the form of either physical or chemically cross-linked hydrogels. Cross-linkers interact with the amine groups in the chitosan, forming molecular bridges in the chitosan chains. As novel chitosan and carrageenan nanoparticles-based gels showed a good potential for the sustained release of drugs in topical administration , more reterature . When combining chitosan and BTC, gelation occurs at room temperature, and these gels can be used for encapsulating hydrophilic and lipophilic drugs for controlled or sustained drug release. In previously reported procedures, porous chitosan was prepared using BTC as a cross-linker  and suprViscoelastic materials exhibit a reversible response that depends on the rate of applied load. This mechanism usually occurs in polymeric materials. These materials are time-dependent, which makes them strain-rate sensitive. Viscoelasticity is the property of a material that exhibits both viscous and elastic properties when deformed. When a viscoelastic material is subjected to stress, the response is composed of elastic deformation (which stores energy) and viscous flow (which releases energy). The profile of the frequency sweep data gives the degree of dispersion, whereas interparticle association frequency sweep curves can provide information about the product (gel) behavior during storage and application. At certain frequency, either the elastic or viscous seems dominant which indicates the elastic or viscous nature of the structured material . From GrThe phase angle reveals the material deformation to either solid or liquid. Low phase angle results can be seen in a solid, and a high phase angle corresponds to liquid. The gel point is another critical parameter that determines the dynamic viscoelastic properties of the polymer systems. The sudden change in viscosity of the fluid can be determined using the gelling point. At the gelling point, a solution becomes more resistant to flow due to a loss in fluidity. Winter and Chambon ,51 explaw/w of water in some cases) facilitates the incorporation of hydrophilic drugs. The non-specific distribution of the drug in vivo, which affects non-target cells leading to side effects, can be overcome by the introduction of hydrogels to release drugs in a controlled manner [Drugs are released from the polymeric network through four different mechanisms: stimulated release, degradation-controlled, solvent-controlled, and diffusion-controlled release . Thus, td manner ,57. In t9H6O6, Mwt 210.14 g/mol), 5-fluorouracil , dialysis tubing with an average flat width of 25 mm (1.0 inch) and molecular weight cut-off (MWCO) 3500 Da, glacial acetic acid, deuterium chloride, deuterium oxide, and ethanol were all purchased from Sigma-Aldrich, Gillingham (UK). Aluminium specimen stubs (0.5″) and carbon tabs (12 mm diameter) were purchased from Agar Scientific Ltd., Stanstead (UK). Deionised water was used for all preparations.Low molecular weight chitosan ), 1,3,5-benzene tricarboxylic acid . This mixture was heated at 40 °C overnight. BTC solutions were prepared by dissolving BTC powder in ethanol in varying concentrations. Three different concentrations  were, respectively, prepared by mixing: A combination of 1 wt% chitosan solution and 10 mM BTC-ethanol solution (M1);A combination of 1 wt% chitosan solution and 50 mM BTC-ethanol solution (M2);A combination of 0.5 wt% chitosan solution and 50 mM BTC-ethanol solution (M3).Chitosan hydrogels with different concentrations were prepared according to a modification of the method previously reported by Yang et al. . BrieflyThe solutions of chitosan and BTC formed a gel at room temperature upon mixing. A consistent amount of drug (15 mg) was loaded in all formulations to have a final concentration of 0.3 mg/mL of 5-FU.The docking procedure for subsequent ligands was performed using AutoDock Vina (docking software)  and UCSF2O (20%). After dissolution, the chitosan solution was transferred to an NMR tube (5 mm). The experiments were carried out at 70 °C, a temperature at which solvent peaks does not interfere with the chitosan’s peaks. The chemical shifts of the chitosan and BTC protons were recorded using the spectra. The typical proton NMR spectra of chitosan and chitosan–BTC were performed on a Bruker Avance III 400 MHz two-channel FT-NMR spectrometer. An aliquot of lyophilized sample (c.a. 15 mg) was dissolved in DCl/DTM iS5 FTIR spectrophotometer in the range of 4000–400 cm−1 with an accumulation of 16 scans. The sample spectrum was collected and processed using OMNIC thermo scientific software.Hydrogel formulations were analyzed using a thermo-scientific NicoletThe viscosity of the hydrogels was determined at room temperature using a Brookfield dial (DV-II + Pro) viscometer. The formulations were placed in beakers (25 mL), and measurements were taken using an LV spindle 64. The viscosity readings (in cP) were recorded at different shear rates  with the torque range between 10 and 100%. The viscometry parameters were measured at 25 ± 0.05 °C in triplicates. The texture profile analysis (TPA) of the hydrogels was carried out using a TA.XT.Plus texture analyser (Stable Micro System). This instrument can be used to determine the physical characteristics, cohesiveness, adhesiveness, consistency, and firmness of gels or other semisolid dosage forms ,62. The Rheological measurements of the chitosan–BTC hydrogels were carried out using a rotational Malvern Kinexus Pro rheometer using rSpace software. The hydrogels were tested after 7 days of preparation using the probe CP4/40 SR4147SS. Measurements were taken at 37 °C for all samples. The samples were dispensed on the surface of the preheated lower plate, and the upper cone was allowed a gap of 0.1424 mm. The excess hydrogel was removed using a spatula. Then, the temperature was allowed to equilibrate to 37 °C. Once stabilized, the flow curves were recorded with varied shear rates at the same temperature. The tests were performed in triplicates.Oscillation amplitude sweep and frequency sweep tests within the linear viscoelastic region (LVE) were carried out at 37 °C. Strain sweep tests were performed using a strain from 0.1 to 100% to the hydrogel once the gel was allowed to reach equilibrium. These tests measure the storage and loss modulus with respect to shear strain at constant temperature (37 °C) and frequency (1 Hz). In the frequency sweep, the frequency was varied (0.1 to 10%), while the amplitude of the deformation was kept constant. The storage and loss modulus of the hydrogels were plotted against frequency to determine the viscoelastic properties of the gels. The morphology of the hydrogels was analysed using a Zeiss EVO 50 scanning electron microscope (SEM). The hydrogels were freeze-dried and positioned on aluminum specimen stubs on which 12 mm diameter carbon tabs were placed. The gels were coated with gold using a sputter coater (SC7640) and scanned at an extra high-tension voltage of 10–20 kV. The specimen was adjusted to a height of 2 mm with a diameter of 35 mm. The samples were viewed using different magnifications, and the software used for SEM operation was Zeiss Smart SEM.2 = 0.9912). The release of 5-FU was studied at 37 °C. Briefly, samples  were placed in a dialysis bag and kept in phosphate buffered saline (PBS solution) (50 mL) at pH 7.4 . At regup < 0.05 was considered statistically significant. The results from the experimental data were presented as mean ± standard deviation. The error bars in the graph represent the standard deviation (n = 3). The results attained were analyzed using SPSS® statistical software.Statistical differences in the drug release profile were assessed using an ANOVA test , and a v1H NMR analysis of chitosan–BTC was carried out using DCl/D2O [Chitosan hydrogels were prepared by mixing different ratios of chitosan and BTC. Following a visual assessment, no differences in the appearance of the samples (M1–M3) were observed; a picture of M1 is reported in  DCl/D2O . The neuc, 1.98 ppm) as proposed by Shigemasa et al. [The DDA was calculated using integrals of the proton peaks of the deacetylated monomer  and protons of the acetyl group  δ 4.71 ppm , 3.75 ppm , 3.10 ppm , 1.98 ppm .Chitosan (i): 1H NMR  δ 13.52 ppm , 8.63 ppm .BTC (iii): 1H NMR  δ 8.52 ppm , 3.65 ppm , 3.02 ppm , 1.90 ppm .Chitosan glucosamine carboxylate salt (iv): To further elucidate the interaction between the polymer (chitosan) and the cross-linked (BTC), a computational study was conducted. Docking is a computational procedure that helps in predicting the binding of one molecule to the pocket of another molecule . This viREMARK VINA RESULT: −3.3 0.267 4.922In the above-represented docking studies , the ami−1 due to O-H and N-H bending (both overlapping in the same region). The band at 2556 cm−1 corresponds to the bending of the OH group of the carboxylic acid group (BTC), which cannot be seen in the uncross-linked chitosan hydrogel. In addition, weak C-H bending of the aromatic ring of BTC can be seen in the cross-linked gels. The bands at 1357 cm−1 and 1272 cm−1 are due to O-H and C-N bending vibrations. The band at 1429 cm−1 can be seen in both cross-linked and uncross-linked gels. The strong absorption band at 1549 cm−1 corresponds to amide bonds of chitosan. Another band at 1626 cm−1 can be due to amine N-H symmetrical vibration [−1 and further confirms the presence of BTC in the structure. The strong bending at 682, 741, and 899 cm−1 is attributed to aromatic C-H bending, which became less intense at increased concentrations. The C-O-C glycosidic linkage from the chitosan can be seen at 1065 cm−1. The interaction between chitosan and BTC can also be proven from the presence of C-O-C bend in the cross-linked signals. These interpretations prove effective cross-linking of chitosan with BTC that takes place at the amino group of chitosan.The FTIR spectrum of not cross-linked and of cross-linked hydrogels can be seen in ibration . The hydThe effect of the cross-linker on the polymer concentrations are listed in The values for the torque were recorded between 10 and 100%, and those for the viscosity of the hydrogels were recorded at different shear rates (rpm). An increase in shear rate makes the fluid layers slide over one another at high speed and influences the viscosity of the material. The viscosity of all the formulations decreased with an increase in shear rate which confirms the pseudoplastic behavior of the fluids. Pseudoplasticity is a characteristic feature of shear-thinning fluids and is time-independent. Shear thinning behavior of hydrogel systems are great for biomedical applications. The non-Newtonian behavior is related to the structural reformation of molecules due to flow. High shear rates can cause the breakdown of fluid structures leading to reduced viscosity. This property influences the performance of the hydrogel during drug delivery via injection . In addiDuring the test, the probe traveled at a speed of 2 mm/s downwards into the gel and was then withdrawn. Gel parameters, including hardness, cohesiveness, and adhesiveness, were evaluated using a standard force-time plot  images of chitosan–BTC gels demonstrated the presence of interconnected pores between the gel networks as seen in t-test (p = 0.21) indicates no significant difference in time required for drug release. However, there is a significant difference in time for drug release between M2 and M3 (p = 0.007). Based on these studies, the prepared hydrogel can be used for drug delivery systems and other biomedical applications.At pH 6.5, rapid release of the drug occurs from 0 to 5 min, and then the drug is released gradually from 5 to 180 min  release than at pH 6.5. This might suggest that 5-FU is not released via erosion of the hydrogel, ; 5-FU is rather released via diffusion which is favoured in non-acidic environments.The current research aimed to develop BTC-cross-linked chitosan hydrogels containing 5-FU for cancer therapy. Using different ratios of BTC/chitosan, flow curves of the hydrogels revealed shear thinning behavior of the resulting hydrogels. With an increase in the shear rate, there is a decline in the apparent viscosity, which is a characteristic feature of shear-thinning fluids. Viscoelastic investigations of the hydrogels revealed the elastic (G′) modulus values were higher than the viscous (G″) values, hence confirming the elastic behavior of all prepared hydrogels. Further oscillatory tests (strain and frequency sweep) confirmed the stable hydrogels’ behavior since all exhibited a plateau in the range of 0.1–10 Hz. Under physiological conditions, the gel behaviour can be tuned by changing the cross-linker concentrations. The low G′ (77.15 Pa) and G″ (8.711 Pa) values of M2 indicate poor mechanical strength when compared to other samples. Spectroscopic and structural investigations of the formulated hydrogels  confirmed the presence of glycosidic bonds signals in all the spectra. The cross-sectional freeze-dried images exhibited porous, compact, and homogenous distribution of hydrogel networks. This provides a scope for encapsulating drugs in the hydrogel matrix. The hydrogels exhibited slow drug release in PBS (<50%) that provides a scope for sustained drug release. Additionally, the release at slightly acidic pH showed a slower and more prolonged release of the drug, which might be explained in light of the porous structure of the hydrogel matrix, coupled with the ionization of chitosan in acidic pH environments. The drug release also appeared to have a direct correlation with the extent of polymer cross-linking, thus facilitating a sustained drug delivery. This is a desired characteristic of hydrogels which tends to enhance the safety and efficacy of drug therapy. Overall, the data from this study on the properties of hydrogels and their behavior in vitro demonstrates a great potential for enhanced biological performance and thus warrant further investigations.